The invention concerns a method for scanning individual regions with a scanning microscope, the regions of interest being distributed over the entire image field. It is possible to switch rapidly between the individual regions of interest while maintaining the scanning motion. The scanning motion can be accomplished by way of a suitable motion of a scanning mirror.
The invention further concerns an arrangement for beam control in scanning microscopy.
In addition, the invention concerns a scanning microscope that comprises an arrangement for beam control which makes it possible to switch rapidly between the individual regions of interest while maintaining the scanning motion. The scanning microscope can also be configured as a confocal scanning microscope. In particular, in the scanning microscope a light beam produced by an illumination system is guided over a specimen with the interposition of several optical means, and it contains at least one detector that, by way of the several optical means, detects a light proceeding from the specimen.
In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being at right angles to one another so that one mirror deflects in the X and the other in the Y direction. The tilting of the mirrors is brought about, for example, using galvanometer positioning elements; both fast resonant galvanometer positioning elements and slower (more accurate) non-resonant ones are used. In order to scan a sample in the specimen plane, it is important that the rotation axes of the mirror lie in or at least near a plane, also referred to as the Fourier plane, conjugated with the focal plane. One possible beam deflection device that meets the requirements for telecentric imaging is known, for example, from DE 196 54 210. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. Usually the positioning elements are equipped with sensors for ascertaining the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the excitation stop), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused on the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, thus yielding a point datum that results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually obtained by acquiring image data in layers.
Ideally, the track of the scanning light beam on or in the specimen describes a meander that fills the entire image field (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). At high beam deflection speeds, deviations from the ideal track occur because of the inertia of the deflecting moving parts, for example the galvanometer shaft and the mirrors. At usable scanning rates ( greater than 100 Hz) the scanning track of the light beam actually describes a sine curve, which in fact a necessitates a correction of the deviations from the ideal situation resulting therefrom.
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus scanned one grid point at a time. The reading must be unequivocally associated with the pertinent scan position so that an image can be generated from the measured data. Advantageously this is done by also continuously measuring the status data of the adjusting elements of the beam deflection device, or (although this is less accurate) by directly using the reference control data of the beam deflection device.
In some microscopy applications the user is interested only in information about individual regions within the image field, while the surrounding sample regions are not of interest. The regions of interest should moreover be scanned as quickly as possible in succession.
Known arrangements offer only a limited capability for scanning individual sample regions of interest. Scanning the entire image field and subsequently selecting the data of the regions of interest is feasible, if at all, only to a limited extent given the required rapid sequential acquisition of information about the regions of interest.
The approach of sequentially scanning the individual regions of interest is better. It is possible in principle to activate the beam deflection device in such a way that each of the regions of interest is separately scanned, for example, in meander fashion, and the surrounding regions that are not of interest are not scanned. This procedure is possible, however, only if the beam deflection device allows the scanning light beam to be specifically controlled and specifically directed onto individual points in the image field.
This is not possible when using resonantly operating beam deflection devices which are based, for example, on the use of resonant galvanometers or micromirrors, because these beam deflection devices operate exclusively at the particular resonant frequency dictated by their design. It is not possible to xe2x80x9cparkxe2x80x9d the light beam in one region of the image field. Difficulties also occur with rapidly deflecting non-resonantly operating beam deflection devices in terms of the positionability that can be achieved, since the positioning elements react to an activation signal in delayed fashion because of their inertia.
It is therefore the object of the invention to describe a method for scanning microscopic preparations with a light beam that solves the problem described above.
This object is achieved by way of a method that comprises the following steps:
acquiring a preview image;
marking at least one region of interest in the preview image;
displacing a scan field onto the region of interest by means of a first beam deflection device; and
acquiring an image by meander-shaped scanning of the region of interest with a second beam deflection device.
What has been recognized according to the present invention is that it is not necessary to forgo the use of fast or resonant beam deflection devices if the scan field swept by the first beam deflection device is displaced within the image field onto the regions of interest with the aid of a further suitable beam deflection device that allows exact positioning.
A further object of the invention is to create an arrangement for beam control which makes it possible to switch rapidly among several regions of interest and, in that context, to collect information from regions of interest based on a consistent pattern.
This object is achieved by an arrangement for beam control in a scanning microscope. The arrangement comprises:
a scanning microscope defining a scan field;
means for acquiring and displaying a preview image
a microscope optical system;
means for marking at least one region of interest in the preview image;
a first beam deflection device for displacing the scan field onto the region of interest; and
a second beam deflection device for meander-shaped scanning within the scan field.
In a particular embodiment, according to the present invention an imaging optical system is provided between the beam deflection devices in order to guarantee the principle of telecentric scanning.
A further object of the invention is to create a scanning microscope that makes possible rapid sequential scanning of sample regions of interest.
This object is achieved by a scanning microscope which comprises:
an arrangement for beam control,
means for acquiring and displaying a preview image
a microscope optical system,
means for marking at least one region of interest in the preview image,
a first beam deflection device for displacing the scan field onto the region of interest; and
a second beam deflection device for meander-shaped scanning within the scan field.
The invention has the advantage that it is not necessary to forgo the use of fast or resonant beam deflection devices if the scan field swept by the first beam deflection device is displaced within the image field onto the regions of interest with the aid of a second suitable beam deflection device that allows exact positioning. Ideally, the track of the scanning light beam on or in the specimen describes a rectangular curve (scan one line in the X direction at a constant Y position, then stop the X scan and slew by Y displacement to the next line to be scanned, then scan that line in the negative X direction at constant Y position, etc.). At increasingly high deflection speeds, the scanning track deviates more and more from the rectangular shape. This phenomenon is attributable substantially to the inertia of the moving elements. With rapid scanning, the scanning track is more similar to a sine curve. In this context, the scan field is swept by the light beam in such a way that the reversing points of the sinusoidal track lie outside the region of interest. The scanning light beam thus describes approximately straight tracks on the region of interest.